Amphoteric liposomes

ABSTRACT

A serum-stable mixture of lipids capable of encapsulating an active agent to form a liposome, said mixture comprising phosphatidylcholine and phosphatidylethanolamine in a ratio in the range of about 0.5 to about 8. The mixture may also include pH sensitive anionic and cationic amphiphiles, such that the mixture is amphoteric, being negatively charged or neutral at pH 7.4 and positively charged at pH 4. Amphoteric liposomes comprising such a mixture may be used for encapsulating nucleic acid therapeutics, such as oligonucleotides and DNA plasmids. The drug/lipid ratio may be adjusted to target the liposomes to particular organs or other sites in the body.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/521,857, filed on Sep. 15, 2006 now abandoned, which claims priorityfrom U.S. Provisional Application Ser. No. 60/717,199, filed on Sep. 15,2005; U.S. Provisional Application Ser. No. 60/717,291, filed on Sep.15, 2005; U.S. Provisional Application Ser. No. 60/717,293, filed Sep.15, 2005; European Application No. 05020218.3, filed on Sep. 15, 2005;European Application No. 05020217.5, filed on Sep. 15, 2005; EuropeanPatent Application No. 050202167.7, filed on Sep. 15, 2005; PCTApplication No. PCT/EP2005/011905, filed on Nov. 4, 2005; PCTApplication No. PCT/EP2005/011908, filed on Nov. 4, 2005; U.S.application Ser. No. 11/266,999, filed on Nov. 4, 2005 (abandoned); U.S.application Ser. No. 11/267,423, filed on Nov. 4, 2005 (abandoned);European Application No. 06113784.0, filed on May 10, 2006; and EuropeanApplication No. 05090322.8, filed on Nov. 21, 2005. The contents of eachof these applications are incorporated herein by reference in theirentireties.

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the U.S. and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference. More generally, documents orreferences are cited in this text, either in a Reference List before theclaims, or in the text itself; and, each of these documents orreferences (“herein-cited references”), as well as each document orreference cited in each of the herein-cited references (including anymanufacturer's specifications, instructions, etc.), is hereby expresslyincorporated herein by reference. Documents incorporated by referenceinto this text may be employed in the practice of the invention.

FIELD OF THE INVENTION

The present invention relates to amphoteric liposomes and has particularreference to such liposomes having improved stability in human or animalserum. The present invention also comprehends mixtures of lipids capableof encapsulating active agents or ingredients such, for example, asdrugs to form liposomes and pharmaceutical compositions comprising suchliposomes.

BACKGROUND OF THE INVENTION

Oligonucleotides represent a novel class of drugs that can veryspecifically down-regulate or interfere with protein expression. Sucholigonucleotides include antisense, locked nucleic acids (LNA), peptidenucleic acids (PNA), morpholino nucleic acids (Morpholinos), smallinterfering RNAs (siRNA) and transcription factors decoys of variouschemistries. A detailed description of the different mechanisms ofaction of such oligonucleotide therapeutics can be found in theliterature (e.g., Crooke in BBA (1999), 1489(1), 31-44; Tijsterman, etal. in Cell (2004), 117(1), 1-3; and Mann, et al. in J Clin Invest,(2000), 106(9), 1071-5).

The use of oligonucleotides for gene repair applications (see, e.g.,Richardson, et al. in Stem Cells (2002), 20, 105-118) and micro RNAs areother examples from this rapidly growing field.

It is known in the art that nucleic acid therapeutics, irrespective oftheir actual chemical origin, may lack therapeutic efficacy owing totheir instability in body fluids or because of inefficient uptake intocells, or both. Chemical modifications of such oligonucleotide,including the above-mentioned variants, as well as the formation ofconjugates with ligands or polymers, represent one strategy to overcomesuch practical limitations.

A second set of strategies involves the use of carrier systems, inparticular liposomes, for protecting, targeting and affording enhanceduptake into cells. Liposomes are artificial single, oligo ormultilamellar vesicles having an aqueous core and being formed fromamphiphilic molecules having both hydrophobic and hydrophilic components(amphiphiles). The cargo may be trapped in the core of the liposome,disposed in the membrane layer or at the membrane surface. Such carriersystems should meet an optimum score of the following criteria: highencapsulation efficiency and economical manufacture, colloidalstability, enhanced uptake into cells and of course low toxicity andimmunogenicity.

Anionic or neutral liposomes are often excellent in terms of colloidalstability, as no aggregation occurs between the carrier and theenvironment. Consequently their biodistribution is excellent and thepotential for irritation and cytotoxicity is low. However, such carrierslack encapsulation efficiency and do not provide an endosomolytic signalthat facilitates further uptake into cells (Journal of Pharmacology andExperimental Therapeutics (2000), 292, 480-488 by Klimuk, et al.).

A great many of publications deal with cationic liposomal systems; see,e.g., Molecular Membrane Biology (1999), 16, 129-140 by Maurer, et al.;BBA (2000) 1464, 251-261 by Meidan, et al.; Reviews in Biology andBiotechnology (2001), 1(2), 27-33 by Fiset & Gounni. Although cationicsystems provide high loading efficiencies, they lack colloidalstability, in particular after contact with body fluids. Ionicinteractions with proteins and/or other biopolymers lead to in situaggregate formation with the extracellular matrix or with cell surfaces.Cationic lipids have often been found to be toxic as shown by Filion, etal. in BBA (1997), 1329(2), 345-356; Dass in J. Pharm. Pharmacal.(2002), 54(5), 593-601; Hirko, et al. in Curr. Med. Chem., 10(14),1185-1193.

These limitations were overcome by the addition of components thatprovide a steric stabilisation to the carriers. Polyethylenglycols ofvarious chain length, for example, are known to eliminate aggregationproblems associated with the use of cationic components in body fluids,and PEGylated cationic liposomes show enhanced circulation times in vivo(BBA (2001) 1510, 152-166 by Semple, et al.). However, the use of PEGdoes not solve the intrinsic toxicity problems associated with cationiclipids. It is also known that PEG substantially inhibits the productiveentry of such liposomes into the cells or their intracellular delivery(Song, et al. in BBA (2002), 1558(1), 1-13). Quite recently, Morrissey,et al. (Nature Biotechnology (2005), 23 (8), 1002-1007) described adiffusible PEG-lipid for a cationic vector that is able to transfersiRNA into liver cells in vivo. However, the huge demand for suchsolutions and the given attrition rate of clinical development more thanmotivates the development of conceptually independent solutions.

Amphoteric liposomes represent a recently described class of liposomeshaving an anionic or neutral charge at pH 7.5 and a cationic charge atpH 4. WO 02/066490, WO 02/066012 and WO 03/070735, all to Panzner, etal. and incorporated herein by reference, give a detailed description ofamphoteric liposomes and suitable lipids therefor. Further disclosuresare made in WO 03/070220 and WO 03/070735, also to Panzner, et al. andincorporated herein by reference, which describe further pH sensitivelipids for the manufacture of such amphoteric liposomes.

Amphoteric liposomes have an excellent biodistribution and are very welltolerated in animals. They can encapsulate nucleic acid molecules withhigh efficiency.

The use of amphoteric liposomes as carriers for drugs for the preventionor treatment of different conditions or diseases in mammals requiresstability of the liposomes after their injection into the bloodstream.For systemic applications especially, the drug must be stablyencapsulated in the liposomes until eventual uptake in the target tissueor cells. The FDA's guidelines prescribe specific preclinical tests fordrugs comprising liposomal formulations. For example, the ratio ofencapsulated drug to free drug must be determined during the circulationtime in the bloodstream.

After the injection of liposomes into the bloodstream, serum componentsinteract with the liposomes and may lead to permeabilisation of theliposomal membrane. However, the release of a drug that is encapsulatedby the liposome also depends upon the molecular dimensions of the drug.This means that a plasmid drug with a size of thousands of base pairs,for example, may be released much more slowly than smalleroligonucleotides or other small molecules. For liposomal delivery ofdrugs it is essential that the release of the drug during thecirculation of the liposomes is as low as possible.

OBJECTS OF THE INVENTION

An object of the present invention therefore is to provide liposomes andmixtures of lipids capable of forming such liposomes having improvedstability upon contact with human or animal serum.

In particular, an object of the present invention is to provideamphoteric liposomes having such improved serum stability.

Another object of the invention is to provide pharmaceuticalcompositions comprising such liposomes as a carrier for the targeteddelivery of active agents or ingredients, including drugs such asnucleic acid drugs, e.g., oligonucleotides and plasmids.

A particular object of the present invention is to provide such apharmaceutical composition for the treatment or prophylaxis ofinflammatory, immune or autoimmune disorders of humans or non-humananimals.

Yet another object of the present invention is to provide methods forthe treatment of human or non-human animals in which a pharmaceuticalcomposition comprising an active agent is targeted to a specific organor organs, tumours or sites of infection or inflammation.

SUMMARY OF THE INVENTION

According to one aspect of the present invention therefore there isprovided a mixture of lipids capable of encapsulating an active agent toform a liposome, said mixture comprising phosphatidylcholine (PC) andphosphatidylethanolamine (PE) in a ratio of phosphatidylethanolamine tophosphatidylcholine in the range of about 0.5 to about 8.

Suitably, said ratio range from about 0.75 to about 5, preferably fromabout 1 to about 4.

In some embodiments, said phosphatidylcholine may be selected from DMPC,DPPC, DSPC, POPC or DOPC, or from phosphatidylcholines from naturalsources such as, for example, soy bean PC and egg PC.

Said phosphatidylethanolamines may be selected from DOPE, DMPE and DPPE.

Preferred neutral lipids include DOPE, POPC, soy bean PC and egg PC.

It is known that cholesterol may stabilise phosphatidylcholine bilayersagainst serum attack. However, neither POPC nor DOPE form serum stablestructures by themselves. It has now been found surprisingly thatmixtures of DOPE and POPC may form serum stable liposomes.

Accordingly, in a particular aspect of the present invention, saidmixture of lipids may be neutral. In some embodiments said mixture mayconsist or consist essentially of phosphatidylcholine andphosphatidylethanolamine in a ratio in the aforementioned range.

In another aspect of the present invention there are provided neutralliposomes comprising a mixture of lipids in accordance with theinvention. Such liposomes may be used as a serum-stable excipient orcarrier for active agents such as drugs.

In a different aspect of the present invention however, said mixture mayfurther comprise one or more charged amphiphiles.

Preferably said one or more charged amphiphiles are amphoteric, beingnegatively charged or neutral at pH 7.4 and positively charged at pH 4.

By “amphoteric” herein is meant a substance, a mixture of substances ora supra-molecular complex (e.g., a liposome) comprising charged groupsof both anionic and cationic character wherein:

-   -   (i) at least one of the charged groups has a pK between 4 and 8,    -   (ii) the cationic charge prevails at pH 4, and    -   (iii) the anionic charge prevails at pH 8,        resulting in an isoelectric point of neutral net charge between        pH 4 and pH 8. Amphoteric character is by this definition        different from zwitterionic character, as zwitterions do not        have a pK in the range mentioned above. In consequence,        zwitterions are essentially neutrally charged over a range of pH        values; phosphatidylcholines and phosphatidylethanolamines are        neutral lipids with zwitterionic character.

Suitably therefore, said mixture may comprise a plurality of chargedamphiphiles which in combination with one another have amphotericcharacter. Preferably said one or more charged amphiphiles comprise a pHsensitive anionic lipid and a pH sensitive cationic lipid. Herein, sucha combination of a chargeable cation and chargeable anion is referred toas an “amphoteric II” lipid pair. Said chargeable cation may have a pKvalue of between about 4 and about 8, preferably between about 5.0 or5.5 and about 7.0 or 7.5. Said chargeable anion may have a pK value ofbetween about 3.5 and about 7, preferably between about 4 or 4.5 andabout 6.0 or 6.5. Examples include MoChol/CHEMS, DPIM/CHEMS andDPIM/DGSucc.

An “amphoteric I” lipid pair comprises a stable cation (e.g.,DDAB/CHEMS, DOTAP/CHEMS and DOTAP/DOPS) and a chargeable anion, while an“amphoteric III” lipid pair comprises a stable anion and a chargeablecation (e.g., MoChol/DOPG and MoChol/Chol-SO₄).

It is of course possible within the scope of the present invention touse amphiphiles with multiple charges, such as, for example, amphipathicdicarboxylic acids, phosphatidic acid, amphipathic piperazinederivatives and the like. Such multi-charged amphiphiles may be pHsensitive amphiphiles or stable anions or cations, or they may have“mixed” character.

Suitably, said anionic lipid may be selected from DOGSucc, POGSucc,DMGSucc, DPGSucc and CHEMS.

Said cationic lipid may be selected from MoChol, HisChol and CHIM.

In yet another aspect of the present invention there are providedamphoteric liposomes comprising phosphatidylcholine andphosphatidylethanolamine in a ratio in the aforementioned range, a pHsensitive anionic lipid and a pH sensitive cationic lipid.

Said amphoteric liposomes may be negatively or neutrally charged at pH7.4 and cationic at pH 4.

In another particular aspect of the present invention, said liposomesencapsulate at least one active agent. Said active agent may comprise adrug. In some embodiments said active agent may comprises a nucleic acidsuch as, for example, an oligonucleotide or DNA plasmid that is capableof being transcribed in a vertebrate cell into one or more RNAs, saidRNAs being mRNAs, shRNAs, miRNAs or ribozymes, said mRNAs coding for oneor more proteins or polypeptides.

Said oligonucleotide or other nucleic acid based drug may beencapsulated in said amphoteric liposomes. A substantial portion or allof said oligonucleotides may be physically entrapped in the amphotericliposomes. The serum stable amphoteric liposomal formulations can beused for the intracellular delivery of drugs or for the prevention ortreatment of a condition and/or disease in mammals or part of mammals,especially humans or their organs.

In some embodiments, said oligonucleotide may be adapted to target anucleic acid encoding CD40, thereby to modulate expression of CD40 inmammalian cells. Suitably, said oligonucleotide may be directed againstthe mRNA of CD40.

In yet another aspect of the present invention there is provided apharmaceutical composition comprising active agent-loaded amphotericliposomes in accordance with the present invention and apharmaceutically acceptable vehicle therefor.

Said composition may be formulated for high or low lipid doses, andsuitably therefore the drug/lipid ratio may be adjusted to a desiredlipid concentration. In some embodiments, said composition may furthercomprise empty liposomes to decrease said drug/lipid ratio, said emptyliposomes having the same or similar size and composition to said activeagent loaded liposomes. Said empty liposomes may comprise a mixture oflipids according to the present invention.

In yet another aspect, the present invention comprehends the use of apharmaceutical composition according to the present invention for theprevention or treatment of an inflammatory, immune or autoimmunedisorder of a human or non-human animal, wherein said compositioncomprises an oligonucleotide adapted to target a nucleic acid encodingCD40 for modulating the expression of CD40 in mammalian cells.

Said composition may be formulated for systemic or local administration.When used systemically, the present invention comprises the use of saidcomposition inter alia for the prevention or treatment of graftrejection, graft-versus-host disease, diabetes type I, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis, asthma,inflammatory bowel disease, psoriasis or thyroiditis.

When formulated for local application, the invention comprises the useof said composition inter alia for the prevention or treatment of graftrejection, graft-versus-host disease, inflammatory bowel disease,asthma, Crohn's disease or ulcerative colitis.

These and other embodiments are disclosed or are obvious from andencompassed by the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying Figures, incorporatedherein by reference, in which:

FIG. 1 is a graph of carboxyfluorescein (CF) release from theMoChol/CHEMS formulations of Table 1 (i.e., formulations comprising 100%DOPE (no POPC), DOPE/POPC ratios of 5, 3, 1.5, and 0.75, and 100% POPC(no DOPE)) below after incubation in full human serum for 4 hours. CFrelease is expressed as % of the unquenched CF signal. The x-axis showsthe total amount of charged lipid at a 1:1 ratio between MoChol andCHEMS.

FIG. 2 is a graph of CF release from the MoChol/DMGSucc formulations ofTable 4 (i.e., formulations comprising DOPE/POPC ratios of 3, 1.5, and0.75) below after incubation in full human serum for 4 hours. CF releaseis expressed as % of the unquenched CF signal. The x-axis shows totalamount of charged lipid at a 1:1 ratio between MoChol and DMGSucc.

FIG. 3 is graph of CF release from liposomes containing MoChol/CHEMS orMoChol/DMGSucc after incubation in full human serum at 37° C. CF releaseis expressed as % of the unquenched CF signal. Excess cation stabilisesthe liposomes against serum attack. DMGSucc is notably more stable thenthe CHEMS counterpart.

FIG. 4 is a graph of CF release from the MoChol/CHEMS and MoChol/DMGSuccformulations of Tables 3 and 6 below after incubation in full humanserum at 37° C. The formulations have DOPE/POPC ratios of 2 and 4 andthe ratio of cationic to anionic lipids is less than 1. Release isexpressed as % of the unquenched CF signal.

FIG. 5 is a bar chart showing the biodistribution of the formulationPOPC/DOPE/MoChol/CHEMS 15:45:20:20 having a size >150 nm whenadministered at low and high lipid doses in rat liver and spleen (seeExample 7 below).

FIG. 6 is a bar chart showing the biodistribution of the formulationPOPC/DOPE/MoChol/CHEMS 15:45:20:20 having a size <150 nm whenadministered at low and high lipid doses in rat liver and spleen (seeExample 7 below).

FIG. 7 is a set of photographs of the limbs of sacrificedcollagen-induced arthritic mice obtained by NIR-imaging and showing thebiodistribution of amphoteric liposomes encapsulating Cy5.5 labelledCD40 antisense (see Example 8 below).

FIG. 8 is a graph showing the effect of treatment with amphotericliposomes containing CD40 antisense on the paw swelling of inflamedmice.

FIG. 9 is a graph of the assessed clinical score of mice treated withamphoteric liposomes containing CD40 antisense.

FIG. 10 is a porcine CD40 cDNA sequence (SEQ ID NO:4) for targeting inaccordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although a number of methodsand materials similar or equivalent to those described herein can beused in the practice of the present invention, the preferred materialsand methods are described herein.

As mentioned above, the amphoteric liposomes of the present inventionmay comprise anionic and cationic components, wherein both componentsare pH-sensitive, as disclosed in WO 02/066012, the contents of whichare incorporated herein by reference.

Cationic lipids that are sensitive to pH are disclosed in WO 02/066489and WO 03/070220, and in the references made therein, in particularBudker, et al. 1996, Nat Biotechnol. 14(6):760-4, the contents of all ofwhich are incorporated herein by reference.

Preferred cationic components are MoChol, HisChol and CHIM, especiallyMoChol.

Preferred anionic lipids are selected from the group comprising:DOGSucc, POGSucc, DMGSucc, DPGSucc and CHEMS, especially DOGSucc,DMGSucc and CHEMS.

The following abbreviations for lipids are used herein, the majority ofwhich abbreviations are in standard use in the literature:

-   -   PC Phosphatidylcholine, unspecified membrane anchor    -   PE Phosphatidylethanolamine, unspecified membrane anchor    -   DMPC Dimyristoylphosphatidylcholine    -   DPPC Dipalmitoylphosphatidylcholine    -   DSPC Distearoylphosphatidylcholine    -   POPC Palmitoyl-oleoylphosphatidylcholine    -   DOPC Dioleoylphosphatidylcholine    -   DOPE Dioleoylphosphatidylethanolamine    -   DMPE Dimyristoylphosphatidylethanolamine    -   DPPE Dipalmitoylphosphatidylethanolamine    -   CHEMS Cholesterolhemisuccinate    -   CHIM Cholesterol-(3-imidazol-1-yl propyl)carbamate    -   DDAB Dimethyldioctadecylammonium bromide    -   DOTAP (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium salt    -   DOPS Dioleoylphosphatidylserine    -   DOPG Dioleoylphosphatidylglycerol    -   Chol-SO₄ cholesterol sulfate    -   MoChol 4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate:

-   -   HisChol Histaminyl-Cholesterolhemisuccinate:

-   -   DGSucc 1,2-Dipalmitoyglycerol-3-hemisuccinate (& Distearoyl-,        dimyristoyl-Dioleoyl or palmitoyl-oleoylderivatives) (in the        structure below the acyl chain is shown schematically)

It has been found that the ratio between the cationic and anionic lipids(the charge ratio) not only determines the isoelectric point, but mayalso affect the serum stability of the composition. Accordingly, saidcharge ratio may vary from 4:1 to 1:4, preferably between 3:1 and 1:3(cation:anion).

In some embodiments of the invention, the cation may be present inexcess over the anion. Preferably said charge ratio is between 3:1 and2:1. The total amount of charged lipids may vary from 5 to 95 mol. % ofthe lipid mixture, preferably from 30 to 80 mol. %, and more preferablyfrom 45 or 50 mol. % to 75 mol. %, with the remaining lipids beingformed from the neutral phospholipids PC and PE.

Alternatively, the cation and anion may be present in substantiallyequal amounts. The total amount of charged lipids may vary from 5 to 75mol. % of the lipid mixture, preferably from 20 to 65 mol. %, with theremaining lipids being formed from the neutral phospholipids PC and PE.

In another alternative, the anion may be present in excess over thecation. Said charge ratio may be between 1:3 and 1:2, preferably about1:2 (cation:anion). The total amount of charged lipids may vary from 40mol. % to 75 or 80 mol. % of the lipid mixture, preferably from 45 or 50mol. % to 70 or 75 mol. %, with the remaining lipids being formed fromthe neutral phospholipids PC and PE.

A number of different combinations of cations and anions may be selectedfrom the lists of suitable components given above. Advantageously, theinvention may be practised using MoChol or CHIM as a chargeable cationand CHEMS, DMGSucc or DOGSucc as a chargeable anion.

Presently preferred liposomes are made from a mixture of lipidscomprising POPC and DOPE in a ratio between 1:1 and 1:4 and anamphoteric lipid pair selected from MoChol and CHEMS, MoChol andDMGSucc, MoChol and DOGSucc, CHIM and CHEMS, and CHIM and DMGSucc, in aratio between 3:1 and 1:1, wherein the amount of charged lipids isbetween 30 and 80 mol. % of the lipid mixture.

Specific examples of such liposomes in accordance with the presentinvention include, but are not limited to:

POPC/DOPE/MoChol/CHEMS 6:24:53:17 POPC/DOPE/MoChol/CHEMS 6:24:47:23POPC/DOPE/MoChol/CHEMS 15:45:20:20 POPC/DOPE/MoChol/CHEMS 10:30:30:30POPC/DOPE/MoChol/CHEMS 24.5:35.5:20:20 POPC/DOPE/MoChol/CHEMS16:24:30:30 POPC/DOPE/MoChol/DMGSucc 6:24:53:17 POPC/DOPE/MoChol/DMGSucc6:24:47:23 POPC/DOPE/MoChol/DMGSucc 15:45:20:20 POPC/DOPE/MoChol/DMGSucc10:30:30:30 POPC/DOPE/MoChol/DMGSucc 24.5:35.5:20:20POPC/DOPE/MoChol/DMGSucc 16:24:30:30 POPC/DOPE/MoChol/DOGSucc12.5:37.5:33:17 POPC/DOPE/MoChol/DOGSucc 7.5:22.5:47:23POPC/DOPE/CHIM/CHEMS 12.5:37.5:33:17 POPC/DOPE/CHIM/CHEMS 7.5:22.5:47:23POPC/DOPE/CHIM/DMGSucc 12.5:37.5:33:17 POPC/DOPE/CHIM/DMGSucc7.5:22.5:47:23

Further presently preferred liposomes comprise a mixture of lipidscomprising POPC and DOPE in a ratio between 1:1 and 1:4, DMGSucc orDOGSucc, and MoChol, wherein the molar amount of DMGSucc or DOGSuccexceeds the molar amount of MoChol and the amount of charged lipids isbetween 30 and 80 mol. %. Preferably, the charge ratio is between 1:2and 1:3 and charged components constitute between 45 or 50 mol. % and 70or 75 mol. % of the lipid mixture.

Specific examples of such further liposomes include, but are not limitedto:

POPC/DOPE/MoChol/DMGSucc 6:24:23:47 POPC/DOPE/MoChol/DMGSucc 8:32:20:40POPC/DOPE/MoChol/DMGSucc 10:40:17:33 POPC/DOPE/MoChol/DMG Succ10:20:23:47 POPC/DOPE/MoChol/DMGSucc 13:27:20:40POPC/DOPE/MoChol/DMGSucc 10:30:20:40 POPC/DOPE/MoChol/DMGSucc17:33:17:33 POPC/DOPE/MoChol/DOGSucc 12.5:37.5:17:33

Without being limited to such use, the materials described in thepresent invention are well suited for use as carriers for nucleicacid-based drugs such as for example oligonucleotides and DNA plasmids.These drugs are classified into nucleic acids that encode one or morespecific sequences for proteins, polypeptides or RNAs and intooligonucleotides that can specifically regulate protein expressionlevels or affect the protein structure through inter alia interferencewith splicing and artificial truncation.

In some embodiments of the present invention, therefore, the nucleicacid-based therapeutic may comprise a nucleic acid that is capable ofbeing transcribed in a vertebrate cell into one or more RNAs, which RNAsmay be mRNAs, shRNAs, miRNAs or ribozymes, wherein such mRNAs code forone or more proteins or polypeptides. Such nucleic acid therapeutics maybe circular DNA plasmids, linear DNA constructs, like MIDGE vectors(Minimalistic Immunogenically Defined Gene Expression) as disclosed inWO 98/21322 or DE 19753182, or mRNAs ready for translation (e.g., EP1392341).

In another embodiment of the invention, oligonucleotides may be usedthat can target existing intracellular nucleic acids or proteins. Saidnucleic acids may code for a specific gene, such that saidoligonucleotide is adapted to attenuate or modulate transcription,modify the processing of the transcript or otherwise interfere with theexpression of the protein. The term “target nucleic acid” encompassesDNA encoding a specific gene, as well as all RNAs derived from such DNA,being pre-mRNA or mRNA. A specific hybridisation between the targetnucleic acid and one or more oligonucleotides directed against suchsequences may result in an inhibition or modulation of proteinexpression. To achieve such specific targeting, the oligonucleotideshould suitably comprise a continuous stretch of nucleotides that issubstantially complementary to the sequence of the target nucleic acid.

Oligonucleotides fulfilling the abovementioned criteria may be builtwith a number of different chemistries and topologies. Oligonucleotidesmay be single stranded or double stranded.

The mechanisms of action of oligonucleotides may vary and might compriseeffects on inter alfa splicing, transcription, nuclear-cytoplasmictransport and translation.

In a preferred embodiment of the invention single strandedoligonucleotides may be used, including, but not limited to, DNA-basedoligonucleotides, locked nucleic acids, 2′-modified oligonucleotides andothers, commonly known as antisense oligonucleotides. Backbone or baseor sugar modifications may include, but are not limited to,Phosphothioate DNA (PTO), 2′O-methyl RNA (2′Ome), 2′ O-methoxyethyl-RNA(2′MOE), peptide nucleic acids (PNA), N3′-P5′ phosphoamidates (NP),2′fluoroarabino nucleic acids (FANA), locked nucleic acids (LNA),Morpholine phosphoamidate (Morpholino), Cyclohexene nucleic acid (CeNA),tricyclo-DNA (tcDNA) and others. Moreover, mixed chemistries are knownin the art, being constructed from more than a single nucleotide speciesas copolymers, block-copolymers or gapmers or in other arrangements. Inaddition to the aforementioned oligonucleotides, protein expression canalso be inhibited using double stranded RNA molecules containing thecomplementary sequence motifs. Such RNA molecules are known as siRNAmolecules in the art (e.g., WO 99/32619 or WO 02/055693). Again, variouschemistries were adapted to this class of oligonucleotides. Also,DNA/RNA hybrid systems are known in the art.

In another embodiment of the present invention, decoy oligonucleotidescan be used. These double stranded DNA molecules and chemicalmodifications thereof do not target nucleic acids but transcriptionfactors. This means that decoy oligonucleotides bind sequence-specificDNA-binding proteins and interfere with the transcription (e.g.Cho-Chung, et al. in Curr. Opin. Mol. Ther., 1999).

In a further embodiment of the invention, oligonucleotides that mayinfluence transcription by hybridizing under physiological conditions tothe promoter region of a gene may be used. Again various chemistries mayadapt to this class of oligonucleotides.

In a still further alternative of the invention, DNAzymes may be used.DNAzymes are single-stranded oligonucleotides and chemical modificationsthereof with enzymatic activity. Typical DNAzymes, known as the “10-23”model, are capable of cleaving single stranded RNA at specific sitesunder physiological conditions. The 10-23 model of DNAzymes has acatalytic domain of 15 highly conserved deoxyribonucleotides, flanked by2 substrate-recognition domains complementary to a target sequence onthe RNA. Cleavage of the target mRNAs may result in their destructionand the DNAzymes recycle and cleave multiple substrates.

In yet another embodiment of the invention, ribozymes can be used.Ribozymes are single stranded oligoribonucleotides and chemicalmodifications thereof with enzymatic activity. They can be operationallydivided into two components, a conserved stem-loop structure forming thecatalytic core and flanking sequences which are reverse complementary tosequences surrounding the target site in a given RNA transcript.Flanking sequences may confer specificity and may generally constitute14-16 nt in total, extending on both sides of the target site selected.

In a still further embodiment of the invention, aptamers may be used totarget proteins. Aptamers are macromolecules composed of nucleic acids,such as RNA or DNA, and chemical modifications thereof that bind tightlyto a specific molecular target and are typically 15-60 nt long. Thechain of nucleotides may form intramolecular interactions that fold themolecule into a complex three-dimensional shape. The shape of theaptamer allows it to bind tightly against the surface of its targetmolecule including but not limited to acidic proteins, basic proteins,membrane proteins, transcription factors and enzymes. Binding of aptamermolecules may influence the function of a target molecule.

All of the above-mentioned oligonucleotides may vary in length betweenas little as 10, preferably 15 and even more preferably 18, and 50,preferably 30 and more preferably 25, nucleotides. The fit between theoligonucleotide and the target sequence is preferably perfect with eachbase of the oligonucleotide forming a base pair with its complementarybase on the target nucleic acid over a continuous stretch of theabovementioned number of oligonucleotides. The pair of sequences maycontain one or more mismatches within the said continuous stretch ofbase pairs, although this is less preferred. In general, the type andchemical composition of such nucleic acids is of little impact for theperformance of the inventive liposomes as vehicles be it in vivo or invitro, and the skilled artisan may find other types of oligonucleotidesor nucleic acids suitable for combination with the inventive liposomes.

In a preferred embodiment of the invention however, oligonucleotides mayused that are adapted to target a nucleic acid encoding the CD40 gene,its sense or antisense strand, any exons or introns or untranslatedregions thereof, thereby to modulate expression of CD40 in mammaliancells.

In another preferred embodiment of the invention, said oligonucleotidesmay directed against any mRNA of CD40, wherein such mRNAs includepre-mRNA and their subsequently matured forms.

Protein expression can be specifically down-regulated usingoligonucleotides such, for example, as antisense, locked nucleic acids(LNA), peptide nucleic acids (PNA), morpholino nucleic acids(Morpholinos) and small interfering RNAs (siRNA) of various chemistries.

CD40 was first described by Pauli, et al. 1984 (Cancer Immunol.Immunotherapy 17: 173-179). The protein is primarily expressed ondendritic cells, endothelial cells and B-cells and interacts with itsligand (CD40 ligand or CD154) on T-cells. The signalling between CD40and CD154 is crucial for the development of a humoral immune response.Over-stimulation of the pathway may lead to a variety ofimmune-associated disorders, including graft rejection,graft-versus-host disease, multiple sclerosis, systemic lupuserythematosus, rheumatoid arthritis, asthma, inflammatory bowel disease,psoriasis and thyroiditis. CD40 over-expression might also be involvedin tumour growth (Gruss, et al. 1997, Leuk. Lymphoma. 24(5-6): 393-422)and enhanced levels of a soluble form of CD40 were reported to beassociated with Alzheimers disease (Mocali et al. 2004, Exp Gerontol.39(10):1555-61. CD40 signals into the NF-κB pathway, consequentlyleading to activation of the transcription factor and the eventualrelease of cytokines such as IL-1, TNFα and IFNγ, which in turn activateother cells, thus promoting inflammation using a positive feedbackmechanism.

Inhibition of the early events in the pathway described above has beenproposed as an effective strategy to inhibit immune disorders orinflammation processes. Examples include the competitive binding of TNFαusing antibodies, receptor blocking using antibodies against theTNFα-receptor and competitive inhibition of NF-κB binding. Since CD40signals through its interaction with the trimeric ligand, CD154,inhibition of the signalling event with small molecule inhibitors isunlikely and therapeutic developments have therefore focused on the useof blocking antibodies. More specifically, the CD40/CD154 interactionmay be blocked using antibodies targeted against one of the components,as described by Holstager, et al. 2000 (J. Biol. Chem. 275:15392-15398)or Baccam & Bishop 1999 (Eur. J. Immunol. 29: 3855-3866). However, theCD40 antibodies under development give rise to side reactions, and thereis therefore a need for alternative means to cut the inflammatoryfeedback loop at this point.

A number of oligonucleotide sequences targeted against CD40 mRNA havebeen validated in vitro so far. US 2004/0186071 and U.S. Pat. No.6,197,584, both to Bennett, et al., for example, give a detaileddescription of such oligonucleotides based on antisense mechanisms.Pluvinet, et al. in Blood, 2004 first described the down-regulation ofCD40 using siRNA against the human target. Further, WO 2004/090108 toManoharan describes the applicability of novel oligonucleotides toinhibit the expression of CD40 protein. Indirect means to down-regulatethe CD40 expression are described in DE 10049549 to Hecker and Wagner,using the inhibition of transcription factor IFR-1. Suitable specificnucleic acids for modulating the expression of CD40 are set forth inExample 11 below.

In a particular aspect of the present invention therefore there isprovided a pharmaceutical composition comprising an oligonucleotidedirected against CD40 as an active agent and an amphoteric liposome ofthe present invention as an excipient. Such formulations have been foundto be therapeutically active in the treatment of inflammations andautoimmune disorders, and accordingly the invention further comprehendsthe use of the composition of the invention for the prevention ortreatment of inflammations, immune or autoimmune disorders, includinggraft rejection, graft-versus-host disease, multiple sclerosis, systemiclupus erythematosus, rheumatoid arthritis, asthma, asthma bronchiale,inflammatory bowel disease, psoriasis, thyroiditis, Morbus Crohn,Colitis ulcerosa, COPD and atopic dermatitis.

The pharmaceutical composition of the present invention may also be usedfor topical treatments, for example the treatment of inflamed mucosa. Inparticular, the composition of the invention may be used for thetreatment or prophylaxis of inflammatory bowel disease or graftrejection. The composition of the present invention may also be adaptedfor topical application to the skin or lungs.

Liposomes have been widely used to alter the pharmacokinetic andbiodistribution profile of encapsulated drugs in vivo. The liposomes ofthe present invention, together with their cargo, may be cleared rapidlyand to a great extent by the liver. However, the pharmacokineticparameters as well as the biodistribution pattern may be controlled byadjusting the size of the liposomes and/or the lipid dose as illustratedin the examples below.

In some embodiments, the liposomes of the present invention may have asize greater than about 150 nm. Such liposomes may be administered at alow lipid dose. Said liposomes may be unilamellar, oligolamellar ormultilamellar. Such a dosing scheme allows for effective and rapidtargeting to the liver and avoids the accumulation of liposomes and drugin other organs, such as the spleen.

Alternatively, such liposomes having a size greater than about 150 nmmay be administered at a high lipid dose, leading to saturation of theliver and an alteration of the biodistribution pattern to anaccumulation of the liposomes in the spleen and more distal sites in thecirculation, such as sites of infection or inflammation or tumours.These areas of the body have fenestrated or incomplete capillariesthrough which liposomes may be filtered out. Furthermore, it is knownthat the spleen and such other areas of infection or inflammation andmany tumors often have high contents of macrophages which can remove theliposomes from the circulation.

Said pharmaceutical composition according to the present invention maybe provided with a high lipid dose by different methods. In someembodiments, the drug/lipid ratio of the composition can be lowered toachieve the desired lipid concentration. Alternatively, the lipidconcentration of the pharmaceutical composition may be controlled byadding empty liposomes of comparable composition and size to the drugloaded liposomes.

In some embodiments, the liposomes according to the present inventionmay have a size of less than about 150 nm. Said liposomes may beunilamellar, oligolamellar or multilamellar. The spleen acts as a filterwhich removes unwanted red blood cells and particles from the blood.Large liposomes are also retained by the reticular filter in the sameway. However, small liposomes may escape and thus do not accumulate inspleen. Accordingly, liposomes according to the present invention,having a size of less than 150 nm may circumvent the spleen as an organ.

Such liposomes having a size of less than 150 nm may be administered ata low lipid dose in order to target liver cells. Such liposomes areparticularly well adapted to penetrate fully the entire liver and toreach a substantial portion of the parenchymal cells of the liver suchas hepatocytes.

Alternatively, said liposomes having a size of less than 150 nm may beadministered at a high lipid dose to target more distal sites in thecirculation, such as areas of infection or inflammation or solidtumours, and simultaneously to circumvent the spleen.

In general, the pharmacokinetic profile and the biodistribution of theliposomes of the present invention may depend upon many factors. Next tothe lipid composition of the liposomes, the size and lipid dosedetermine the in vivo fate of the liposomes. The liposomes of theinvention may be unilamellar, oligolamellar or multilamellar,irrespective of their size.

In some embodiments, the liposomes of the present invention may be usedto target an inflamed lung by systemic administration to a human ornon-human animal patient.

Starting from the data presented herein, those skilled in the art willbe able to establish appropriate dosage regimens for other species, inparticular for other mammals or humans. Specifically, whether a lipiddose in another species (e.g. human) is “low” or “high” can bedetermined by pharmacokinetic data. The pharmacokinetic of liposomesfollows a two compartment model. As mentioned above, high lipid doseslead to a saturation of the liver and an alteration of thebiodistribution pattern. This leads to enhanced Cmax values in theterminal part of the pharmacokinetic curve.

The pharmaceutical composition of the present invention may beformulated for use as a colloid in a suitable pharmacologicallyacceptable vehicle. Vehicles such as water, saline, phosphate bufferedsaline and the like are well known to those skilled in the art for thispurpose.

In some embodiments, the composition of the present invention may beadministered at a physiological pH of between about 7 and about 8. Tothis end, the composition comprising the active agent, excipient andvehicle may be formulated to have a pH in this range.

Methods for manufacturing liposomes are known to those skilled in theart. They include, but are not limited to, extrusion through membranesof defined pore size, injection of lipid solutions in ethanol into thewater phase containing cargo or high pressure homogenisation.

Also, it is known in the art that nucleic acid therapeutics can becontacted with the lipids at neutral pH, resulting in volume inclusionof a certain percentage of the solution containing the nucleic acid.High concentrations of lipids ranging from 50 mM to 150 mM are preferredto achieve substantial encapsulation of the drug.

In contrast to such standard procedures, amphoteric liposomes offer thedistinct advantage of binding nucleic acids at or below theirisoelectric point, thereby concentrating the drug at the liposomesurface. Such a process is described in WO 02/066012 in more detail.Upon elevating the pH of the liposomes to physiological pH (about pH7.4) the negatively charged nucleic acids dissociate from the liposomalmembrane. Irrespective of the actual production process, thenon-encapsulated active drug can be removed from the liposomes after theinitial production step, wherein liposomes are formed as tightcontainers. Again, the technical literature and the references includedhere describe such methodology in detail and suitable process steps mayinclude, but are not limited to, size exclusion chromatography,sedimentation, dialysis, ultrafiltration, diafiltration and the like.

In some embodiments of the invention, more than 80 wt. % of the drug maybe disposed inside said liposomes.

However, such removal of non-encapsulated material is not mandatory andin some embodiments the composition may comprises entrapped as well asfree drug.

The particle size of the liposomes may be between 50 and 500 nm,preferably between 50 and 300 nm.

Following is a description by way of example only with reference to theaccompanying drawings of embodiments of the present invention.

EXAMPLES Example 1 Preparation of carboxyfluorescein (CF) LoadedLiposomes with the Amphoteric II Lipids MoChol and CHEMS

Stock solutions of lipids in chloroform were mixed and finallyevaporated in a round bottom flask to dryness under vacuum. Lipid filmswere hydrated with 100 mM CF in PBS pH 7.5. The resulting lipidconcentration was 20 mM. The suspensions were hydrated for 45 minutes ina water bath at room temperature, sonicated for 5 minutes following bythree freeze/thaw cycles at −70° C. After thawing the liposomalsuspensions were extruded 15 times through polycarbonate membranes witha pore size of 100 nm. Non-encapsulated CF was removed by gelfiltration, whereas the liposomes were diluted by a factor of three.Lipid recovery and concentration was analysed by organic phosphateassay. Particle size was measured by dynamic light scattering on aMalvern Zetasizer 3000 HSA.

TABLE 1 Variation of the ratio DOPE/POPC and the total amount of chargedcomponents Lipids Composition DOPE/MoChol/CHEMS 60:20:20DOPE/MoChol/CHEMS 50:20:30 DOPE/MoChol/CHEMS 40:30:30 DOPE/MoChol/CHEMS20:40:40 POPC/MoChol/CHEMS 60:20:20 POPC/MoChol/CHEMS 40:30:30POPC/MoChol/CHEMS 20:40:40 POPC 100 POPC/DOPE 20:80POPC/DOPE/MoChol/CHEMS 10:50:20:20 POPC/DOPE/MoChol/CHEMS 7:35:30:30POPC/DOPE/MoChol/CHEMS 3:17:40:40 POPC/DOPE 25:75 POPC/DOPE/MoChol/CHEMS15:45:20:20 POPC/DOPE/MoChol/CHEMS 10:30:30:30 POPC/DOPE/MoChol/CHEMS5:15:40:40 POPC/DOPE 40:60 POPC/DOPE/MoChol/CHEMS 24.5:35.5:20:20POPC/DOPE/MoChol/CHEMS 16:24:30:30 POPC/DOPE/MoChol/CHEMS 8:12:40:40POPC/DOPE 57:43 POPC/DOPE/MoChol/CHEMS 34:26:20:20POPC/DOPE/MoChol/CHEMS 22.8:17.2:30:30 POPC/DOPE/MoChol/CHEMS11.4:8.6:40:40

TABLE 2 Variation of the ratio MoChol/CHEMS Lipids CompositionPOPC/DOPE/MoChol/CHEMS 6:24:53:17 POPC/DOPE/MoChol/CHEMS 6:24:47:23POPC/DOPE/MoChol/CHEMS 6:24:35:35 POPC/DOPE/MoChol/CHEMS 6:24:23:47

TABLE 3 Variation of ratio DOPE/POPC and the total amount of chargedcomponents Lipids Composition POPC/DOPE/MoChol/CHEMS 4:16:27:53POPC/DOPE/MoChol/CHEMS 6:24:23:47 POPC/DOPE/MoChol/CHEMS 8:32:20:40POPC/DOPE/MoChol/CHEMS 10:40:17:33 POPC/DOPE/MoChol/CHEMS 7:13:27:53POPC/DOPE/MoChol/CHEMS 10:20:23:47 POPC/DOPE/MoChol/CHEMS 13:26:20:40POPC/DOPE/MoChol/CHEMS 17:33:17:33

Example 2 Preparation of carboxyfluorescein (CF) Loaded Liposomes withthe Amphoteric II Lipids MoChol and DMGSucc

Liposomes were prepared as described in Example 1.

TABLE 4 Variation of the ratio DOPE/POPC and the total amount of chargedcomponents Lipids Composition POPC/DOPE/MoChol/DMGSucc 15:45:20:20POPC/DOPE/MoChol/DMGSucc 10:30:30:30 POPC/DOPE/MoChol/DMGSucc 5:15:40:40POPC/DOPE/MoChol/DMGSucc 24.5:35.5:20:20 POPC/DOPE/MoChol/DMGSucc16:24:30:30 POPC/DOPE/MoChol/DMGSucc 8:12:40:40 POPC/DOPE/MoChol/DMGSucc34:26:20:20 POPC/DOPE/MoChol/DMGSucc 22.8:17.2:30:30POPC/DOPE/MoChol/DMGSucc 11.4:8.6:40:40

TABLE 5 Variation of the ratio MoChol/DMGSucc Lipids CompositionPOPC/DOPE/MoChol/DMGSucc 6:24:53:17 POPC/DOPE/MoChol/DMGSucc 6:24:47:23POPC/DOPE/MoChol/DMGSucc 6:24:35:35 POPC/DOPE/MoChol/DMGSucc 6:24:23:47

TABLE 6 Variation of ratio DOPE/POPC and the total amount of chargedcomponents Lipids Composition POPC/DOPE/MoChol/DMGSucc 4:16:27:53POPC/DOPE/MoChol/DMGSucc 6:24:23:47 POPC/DOPE/MoChol/DMGSucc 8:32:20:40POPC/DOPE/MoChol/DMGSucc 10:40:17:33 POPC/DOPE/MoChol/DMGSucc 7:13:27:53POPC/DOPE/MoChol/DMGSucc 10:20:23:47 POPC/DOPE/MoChol/DMGSucc13:26:20:40 POPC/DOPE/MoChol/DMGSucc 17:33:17:33

Example 3 Preparation of carboxyfluorescein (CF) Loaded Liposomes withthe Amphoteric II Lipids MoChol and DOGSucc

Liposomes were prepared as described in Example 1.

TABLE 7 Variation of the ratio MoChol/DOGSucc and the total amount ofcharged components Lipids Composition Serum StabilityPOPC/DOPE/MoChol/DOGSucc 12.5:37.5:17:33 + POPC/DOPE/MoChol/DOGSucc12.5:37.5:33:17 + POPC/DOPE/MoChol/DOGSucc 7.5:22.5:23:47 −POPC/DOPE/MoChol/DOGSucc 7.5:22.5:47:23 +

Example 4 Preparation of carboxyfluorescein (CF) Loaded Liposomes withthe Amphoteric II Lipids CHIM and CHEMS

Liposomes were prepared as described in Example 1.

TABLE 8 Variation of the ratio CHIM/CHEMS and the total amount ofcharged components Serum Lipids Composition StabilityPOPC/DOPE/CHIM/CHEMS 12.5:37.5:17:33 − POPC/DOPE/CHIM/CHEMS12.5:37.5:33:17 + POPC/DOPE/CHIM/CHEMS 7.5:22.5:23:47 −POPC/DOPE/CHIM/CHEMS 7.5:22.5:47:23 +

Example 5 Preparation of carboxyfluorescein (CF) Loaded Liposomes withthe Amphoteric II Lipids CHIM and DMGSucc

Liposomes were prepared as described in Example 1.

TABLE 9 Variation of the ratio CHIM/DMGSucc and the total amount ofcharged components Lipids Composition Serum StabilityPOPC/DOPE/CHIM/DMGSucc 12.5:37.5:17:33 − POPC/DOPE/CHIM/DMGSucc12.5:37.5:33:17 + POPC/DOPE/CHIM/DMGSucc 7.5:22.5:23:47 −POPC/DOPE/CHIM/DMGSucc 7.5:22.5:47:23 +

Example 6 Serum Stability Test of CF-Loaded Amphoteric Liposomes ofExamples 1 and 2

Carboxyfluorescein (CF) was used as model drug to determine the serumstability of amphoteric liposomes. As well as oligonucleotides, CF isnegatively charged.

25 μl of the CF-loaded liposomes were mixed with 100 μl pre-warmed fullhuman serum or PBS, respectively and incubated at 37° C. At defined timepoints 5 μl sample was transferred into a 96-well microliter plate to 20μl PBS, pH 7.5 or 20 μl 20% Triton X-100. Finally 275 μl PBS were addedto each well and fluorescence intensity was measured at 475/530 nm.

The serum stability was observed over a period of 4 hours by determiningthe release of CF from the liposomes via the fluorescence measurement.The released amount of CF (in %) is measured at defined time points aswell as after a treatment of the liposomes with a detergent (TritonX-100) to get a 100% release value.

Results:

Mixtures of POPC and DOPE are stable in serum. POPC itself does not formliposomes that withstand attack from serum. In addition, DOPE does notform liposomes at all. Quite surprisingly, mixtures from both componentswere found to be very stable and resistant against serum attack. In thisexample, DOPE/POPC ratios from 0.75 to 5 were found to form stablestructures with a broad optimum between 1.5 and 5 (see also FIGS. 1 and2).

Charged components and neutral lipids are independent variables. Serumsensitivity for a 1:1 ratio of both MoChol/CHEMS or MoChol/DMGSucc islow to very low and stable particles are formed over a wide range ofmixtures. At least 60 or 70 mol. % of total charged components wasrequired to affect significantly the bilayer stability.

The serum stability of lipid mixtures containing 70% of chargedcomponents (see Tables 2 and 5) is shown in FIG. 3. In general, anexcess of MoChol has a stabilising effect.

The formulations of Tables 3 and 6 that were tested for serum stabilityhave DOPE and POPC in a ratio of either 2:1 or 4:1. The total amount ofthe charged lipids was titrated from 80% down to 50%. The results areshown in FIG. 4.

Example 7 Biodistribution of Serum Stable Amphoteric Liposomes

Stock solutions of lipids (+/−1% 14C-DPPC) in chloroform were mixed andfinally evaporated in a round bottom flask to dryness under vacuum.Lipid films were hydrated with 1.5 ml 3H-Inulin in PBS pH 7.5 or 5 mlPBS alone. The resulting lipid concentration was 100 mM. The suspensionswere hydrated for 45 minutes in a water bath at room temperature,sonicated for 30 minutes following by three freeze/thaw cycles at −70°C. After thawing the liposomal suspensions were extruded 15 timesthrough polycarbonate membranes with an appropriate pore size. Liposomeswere separated from non-encapsulated 3H-Inulin by ultracentrifugation(twice).

Lipid recovery and concentration was analysed by organic phosphate assayand in case of radiolabelled particles, the encapsulation efficiency wasmeasured by liquid scintillation. Particle size was measured by dynamiclight scattering on a Malvern Zetasizer 3000 HSA. The resultingunlabelled and radiolabelled preparations were mixed up and diluted withPBS to the final lipid concentrations.

Formulations:

Size Lipid 3H 14C Number Formulation [nm] [mM] [kBq/ml] [kBq/ml] LD-1POPC/DOPE/MoChol/ 229 12.3 332 52 CHEMS 15:45:20:20 HD-2POPC/DOPE/MoChol/ 231 54.8 453 70 CHEMS 15:45:20:20 LD-3POPC/DOPE/MoChol/ 148 10 173 53 CHEMS 15:45:20:20 HD-4 POPC/DOPE/MoChol/140 50 182 58 CHEMS 15:45:20:20

Biodistribution Study

39 male Wistar rats (Charles River) were divided into five groups andinjected intravenously via the tail vein. At specific time points bloodsamples (for PK) and/or tissue samples (for BD) were collected andanalysed by catalytic oxidation under high temperature. Percentage ofcarry over between samples was determined and included into the analysisof the data set.

Study group Formulation Number Animals 1 POPC/DOPE/MoChol/CHEMS LD-1 915:45:20:20 2 POPC/DOPE/MoChol/CHEMS HD-2 9 15:45:20:20 3POPC/DOPE/MoChol/CHEMS LD-3 9 15:45:20:20 4 POPC/DOPE/MoChol/CHEMS HD-49 15:45:20:20 5 PBS PBS 3

The results of the biodistribution study are shown in FIGS. 5-6 whereinbiodistribution of the different liposomal formulations in liver andspleen is shown. The accumulation of the liposomes in other organs didnot exceed 5% and is therefore not shown. FIG. 5 clearly demonstratesthat amphoteric liposomes of the present invention having a size >150 nmaccumulate solely in the liver when administered in low lipid doses. Incontrast, by administering the same liposomal formulation in a highlipid dose it could be shown that the biodistribution pattern ischanged. Next to the liver the liposomes with a size >150 nm accumulatein spleen as well.

FIG. 6 shows the biodistribution of amphoteric liposomes of the presentinvention prepared in a size <150 nm. Whereas the biodistribution ofthese liposomes administered at low lipid dose does not differ from theliposomes with a size >150 nm, it can be demonstrated that anadministration of the liposomes having a size <150 nm in high lipid dosedoes not lead to an accumulation in spleen.

Example 8 Biodistribution of Amphoteric Liposomes Encapsulating Cy5.5Labelled CD40 Antisense in Collagen Induced Arthritic Mice

Stock solutions of lipids in chloroform were mixed and finallyevaporated in a round bottom flask to dryness under vacuum. Lipid filmwas hydrated with Cy5.5 labelled CD40 antisense in 10 mM NaAc, 50 mMNaCl, pH 4.5. The resulting lipid concentration was 20 mM. Thesuspensions were hydrated for 45 minutes in a water bath at 50° C.,sonicated for 5 minutes following by a freeze/thaw cycle at −70° C.After thawing the liposomal suspensions were extruded 19 times through200 nm polycarbonate membranes. After the extrusion process the pH ofthe liposomal suspension was shifted to pH 7.5 by adding 1/10 Vol. 1MHEPES, pH 8. Non-encapsulated Cy5.5 labelled CD40 antisense was removedby high speed sedimentation (twice) and discarding the supernatant.

Lipid recovery and concentration was analysed by organic phosphateassay. Encapsulation efficiency was measured by fluorescencespectroscopy. Particle size was measured by dynamic light scattering ona Malvern Zetasizer 3000 HSA.

Empty liposomes were produced by injecting 10 Vol-% of an ethanoliclipid solution (a mixture of 15 mol. % POPC, 45 mol. % DOPE, 20 mol. %MoChol and 20 mol. % CHEMS) into 10 mM NaAc 50 mM NaCl pH 4.5. Theresulting lipid concentration was 2 mM. The pH of this solution wasimmediately shifted with 1/10 volume 1M Hepes pH 8. To concentrate thediluted liposomes the suspension was diafiltered.

Encap- Size Lipid sulation Formulation [nm] [mM] Cargo efficiencyPOPC/DOPE/MoChol/CHEMS 192 19 Cy5.5 77% 15:45:20:20 OCD40-DNPOPC/DOPE/MoChol/CHEMS 104 195 empty — 15:45:20:20

For the biodistribution study in mice the filled and empty liposomeswere mixed as follows:

200 μl Cy5.5 liposomes and 41 μl empty liposomes

DBA/1 mice were immunized by subcutaneous injections of type II collagen(200 μg/mouse) emulsified in complete Freund's adjuvant. Mice wereinjected intravenously with the liposomal suspension (241 μl) at day 1of arthritis induction (around day 21 after single immunization withcollagen type II). Day one was defined as the day where the inflammationwas obvious (clinical score after R. O. Williams of at least 2).

Mice were sacrificed ten hours after the injection of the liposomalsuspension. Organs and paws were removed and immediately frozen inliquid nitrogen. The biodistribution of the Cy5.5 labelled CD40antisense encapsulated in the liposomes was assessed by NIR-Imaging andcompared with tissue samples of untreated mice. Specific enrichment wasfound for inflamed paws in mice with active disease. More specifically,accumulation of the amphoteric liposomes coincides with the highlyactive sites of the disease on individual paws or even toes or fingers(see FIG. 7).

Example 9 Preparation of CD40-ODN-Containing Liposomes with the AdvancedLoading Procedure

Liposomes were produced by injecting 10 Vol-% of an ethanolic lipidsolution (a mixture of 15 mol. % POPC, 45 mol. % DOPE, 20 mol. % MoCholand 20 mol. % CHEMS) into 10 mM NaAc 50 mM NaCl pH 4.5 containing 60μg/ml of a 18 bp antisense against CD40.

The resulting lipid concentration was 2 mM. The pH of this solution wasimmediately shifted with 1/10 volume 1M Hepes pH 8. To concentrate thediluted liposomes the suspensions were sedimented for 2 h and 5 min at65,000 rpm at 20° C. in a T865 rotor (Sorvall Ultra Pro 80). Afterwardsthe formulation was sterile filtered through 0.45 μm.

TABLE 9 example for Smarticles formulation which encapsulate CD40 ODNLipid Mol. % size Polydisp. Index POPC/DOPE/MoChol/CHEMS 15:45:20:20178.5 0.317

The amount of encapsulated ODN was measured by checking the opticaldensity (OD) by 260 nm. The following amount of ODN was encapsulated inthe Smarticles formulation.

TABLE 10 encapsulated amount of ODN in the Smarticles formulation μgEncapsu- ODN/μmol lation Lipid Mol. % lipid efficacyPOPC/DOPE/MoChol/CHEMS 15:45:20:20 8.87 29.58%

Example 10 Therapeutic Efficacy in Arthritis

DBA/1 mice were immunized by subcutaneous injections of type II collagen(200 μg/mouse) emulsified in complete Freund's adjuvant. Treatment withSmarticles or controls was initiated at day 1 of arthritis induction(around day 21 after single immunization with collagen type II) andrepeated at day 3 and 5. Day one was defined as the day where theinflammation was obvious (clinical score after R. O. Williams of atleast 2).

For the treatment studies the liposomal CD40-ODN was injectedintravenously into the tail vein of rats with established inflammation.Each dosage contains 4 mg CD40-ODN per kg bodyweight (encapsulatedCD40-ODN).

During the experiment the swelling of paws were observed and theclinical arthritis score were determined.

As evidenced by FIGS. 8 and 9, there was a significant reduction of theswelling of the paws after a treatment with CD40-ODN encapsulated in theamphoteric liposomes. Also the clinical score was significant reducedafter treatment with CD40-ODN encapsulated in such liposomes.

Example 11 Materials

This example provides non-limiting examples of CD40 nucleotide sequencesthat may be targeted by oligonucleotides that modulate the expression ofCD40 and that are suitable for use in the compositions in accordancewith the present invention.

Human CD40 mRNA (GenBank accession no. X60592)

Human CD40 mRNA sequence for targeting in accordance with the presentinvention is presented in SEQ ID NO: 1. Related sequence information isfound in published patent application number US 2004/0186071 (i.e., SEQID NO: 85) to Bennett, et al. and in U.S. Pat. No. 6,197,584 (i.e., SEQID NO: 85) to Bennett, et al. and in Pluvinet, et al., Blood, 2004,104(12), 3642-3646, the contents of which are incorporated by referenceherein.

(SEQ ID NO: 1): 1gcctcgctcg ggcgcccagt ggtcctgccg cctggtctca cctcgccatg gttcgtctgc 61ctctgcagtg cgtcctctgg ggctgcttgc tgaccgctgt ccatccagaa ccacccactg 121catgcagaga aaaacagtac ctaataaaca gtcagtgctg ttctttgtgc cagccaggac 181agaaactggt gagtgactgc acagagttca ctgaaacgga atgccttcct tgcggtgaaa 241gcgaattcct agacacctgg aacagagaga cacactgcca ccagcacaaa tactgcgacc 301ccaacctagg gcttcgggtc cagcagaagg gcacctcaga aacagacacc atctgcacct 361gtgaagaagg ctggcactgt acgagtgagg cctgtgagag ctgtgtcctg caccgctcat 421gctcgcccgg ctttggggtc aagcagattg ctacaggggt ttctgatacc atctgcgagc 481cctgcccagt cggcttcttc tccaatgtgt catctgcttt cgaaaaatgt cacccttgga 541caagctgtga gaccaaagac ctggttgtgc aacaggcagg cacaaacaag actgatgttg 601tctgtggtcc ccaggatcgg ctgagagccc tggtggtgat ccccatcatc ttcgggatcc 661tgtttgccat cctcttggtg ctggtcttta tcaaaaaggt ggccaagaag ccaaccaata 721aggcccccca ccccaagcag gaaccccagg agatcaattt tcccgacgat cttcctggct 781ccaacactgc tgctccagtg caggagactt tacatggatg ccaaccggtc acccaggagg 841atggcaaaga gagtcgcatc tcagtgcagg agagacagtg aggctgcacc cacccaggag 901tgtggccacg tgggcaaaca ggcagttggc cagagagcct ggtgctgctg ctgcaggggt 961gcaggcagaa gcggggagct atgcccagtc agtgccagcc cctc

Mus Musculus CD40 mRNA

Murine CD40 mRNA sequence for targeting in accordance with the presentinvention is presented in SEQ ID NO: 2. Related sequence information isfound in published patent application number US 2004/0186071 (i.e. SEQID NO: 132) to Bennett, et al., the contents of which are incorporatedby reference herein.

(SEQ ID NO: 2):gcctcctggc ccttcagctg tggtctttcc cgttttctga ctttgcggtg acactgggga 60cttccttaga cctctctgga gacgctttcg gttctgcaga gattcccagg ggtattgtgg 120gtggggtggg gtaacaatag tgtccctgtg gcgctcccag tccctatagt aatccttcac 180cectctgcta tcttgcaatc aggagagtcc ttagccctgc tataggtggc ttttgaggtc 240ctggatgcga ggagggggac tggggggtgg gtcgggtaat gtaagaaaag ggctcctttt 300gggaccctgg ctcctccagc caccttggtg cccatccctt aaactcttgg ggacaatcag 360actcctggga aggtcctggg gaaatccctg ctcagtgact agccataggc ccaccgcgat 420tggtgcccga agaccccgcc ctcttcctgg gcgggactcc tagcagggac tttggagtga 480cttgtggctt cagcaggagc cctgtgattt ggctcttctg atctcgccct gcgatggtgt 540ctttgcctcg gctgtgcgcg ctatggggct gcttgttgac agcggtgagt ggcttgtgtt 600ctaacctcca agggagttag ggcttagaga gtgagagatg gaaagaggaa agaggagaca 660agactttgga gatgagagat cttcctactg gaagcggcgg ttagtaggat gggcaagatc 720tctcgcgtct tgacacacac acacacacac acaaatgagg tgggctgctc ctctttcctt 780ccagaaggtc ggggttctgt tccacgaagc ccacagggaa ccttagggag ggcattcctc 840cacagcggtg cctggacagc tttgtctgac ccaagccttg ctccggagct gactgcagag 900actggaaagg gttagcagac aggaagcctg gctggggg 938

Rat CD40 mRNA (GenBank Accession No. AF 241231)

Rat CD40 mRNA sequence for targeting in accordance with the presentinvention is presented in SEQ ID NO: 3. (See, Gao, Ph.D. thesis,Goettingen 2003).

(SEQ ID NO: 3): 1tgggacccct gtgatctggc tgctctgatc tcgctctgca atgctgcctt tgcctcagct 61gtgcgcgctc tggggctgct tgttgacagc ggtccatcta ggacagtgtg ttacgtgcag 121tgacaaacag tacctccaag gtggcgagtg ctgcgatttg tgccagccgg gaaaccgact 181agttagccac tgcacagctc ttgagaagac ccaatgccaa ccgtgcgact caggcgaatt 241ctcagctcac tggaacaggg agatccgctg ccaccagcac cgacactgcg aactcaatca 301agggcttcag gttaagaagg agggcaccgc ggtntcagac actgtttgta cctgcaagga 361agggcagcac tgcgccagca aggagtgcga gacgtgcgct cagcacaggc cctgtggccc 421tggctttgga gtcgtgcaga tggccactga gactactgat accgtctgcc aaccctgccc 481ggtcggattc ttctccaatg ggtcatcact ttttgaaaag tgtcatccat ggacaagctg 541tgaagat

Porcine CD40 cDNA

Porcine CD40 cDNA sequence for targeting in accordance with the presentinvention is presented in SEQ ID NO: 4. (FIG. 10). Related sequenceinformation is found in Rushworth, et al., Transplantation, 2002, 73(4),635-642, the contents of which are incorporated by reference herein.

In addition, the following provide non-limiting examples of anti-CD40oligonucleotides, e.g., antisense CD40 nucleic acid sequences, that aresuitable for use in the present invention:

Oligonucleotides Against Human CD40

Examples of human antisense CD40 oligonucleotides are presented below.Further sequence information is found in published patent applicationnumber US 2004/0186071 and U.S. Pat. No. 6,197,584 to Bennett, et al.,the contents of which are provided by reference herein. The SEQ ID NOS.referred to by Bennett, et al. are provided to the right.

SEQ ID NO: 5 ccaggcggca ggaccact Seq ID No: 1 of Bennett et al.  SEQ ID NO: 6 gaccaggcgg caggacca Seq ID No.: 2 of Bennett et al.SEQ ID NO: 7 aggtgagacc aggcggca Seq ID No: 3 of Bennett et al.SEQ ID NO: 8 gcagaggcag acgaacca Seq ID No: 5 of Bennett et al.SEQ ID NO: 9 gcaagcagcc ccagagga Seq ID No: 6 of Bennett et al.SEQ ID NO: 10 ggtcagcaag cagcccca Seq ID No.: 7 of Bennett et al.SEQ ID NO: 11 gacagcggtc agcaagca Seq ID No: 8 of Bennett et al.SEQ ID NO: 12 gatggacagc ggtcagca Seq ID No: 9 of Bennett et al.SEQ ID NO: 13 tctggatgga cagcggtc Seq ID No: 10 of Bennett et al.SEQ ID NO: 14 ggtggttctg gatggaca Seq ID No: 11 of Bennett et al.SEQ ID NO: 15 gtgggtggtt ctggatgg Seq ID No: 12 of Bennett et al.SEQ ID NO: 16 gcagtgggtg gttctgga Seq ID No: 13 of Bennett et al.SEQ ID NO: 17 ctggcacaaa gaacagca Seq ID No: 15 of Bennett et al.SEQ ID NO: 18 gtgcagtcac tcaccagt Seq ID No: 20 of Bennett et al.SEQ ID NO: 19 attccgtttc agtgaact Seq ID No: 23 of Bennett et al.SEQ ID NO: 20 ttcaccgcaa ggaaggca Seq ID No: 25 of Bennett et al.SEQ ID NO: 21 ctctgttcca ggtgtcta Seq ID No: 26 of Bennett et al.SEQ ID NO: 22 ctggtggcag tgtgtctc Seq ID No: 27 of Bennett et al.SEQ ID NO: 23 ggtgcccttc tgctggac Seq ID No: 31 of Bennett et al.SEQ ID NO: 24 ctgaggtgcc cttctgct Seq ID No: 32 of Bennett et al.SEQ ID NO: 25 gtgtctgttt ctgaggtg Seq ID No: 33 of Bennett et al.SEQ ID NO: 26 acaggtgcag atggtgtc Seq ID No: 35 of Bennett et al.SEQ ID NO: 27 gtgccagcct tcttcaca Seq ID No: 37 of Bennett et al.SEQ ID NO: 28 tgcaggacac agctctca Seq ID No: 40 of Bennett et al.SEQ ID NO: 29 gagcggtgca ggacacag Seq ID No: 41 of Bennett et al.SEQ ID NO: 30 aatctgcttg accccaaa Seq ID No: 43 of Bennett et al.SEQ ID NO: 31 gctcgcagat ggtatcag Seq ID No: 46 of Bennett et al.SEQ ID NO: 32 gcagggctcg cagatggt Seq ID No: 47 of Bennett et al.SEQ ID NO: 33 gactgggcag ggctcgca Seq ID No: 49 of Bennett et al.SEQ ID NO: 34 gcagatgaca cattggag Seq ID No: 52 of Bennett et al.SEQ ID NO: 35 tcgaaagcag atgacaca Seq ID No: 53 of Bennett et al.SEQ ID NO: 36 gtccaagggt gacatttt Seq ID No: 54 of Bennett et al.SEQ ID NO: 37 caggtctttg gtctcaca Seq ID No: 57 of Bennett et al.SEQ ID NO: 38 ctgttgcaca accaggtc Seq ID No: 58 of Bennett et al.SEQ ID NO: 39 gtttgtgcct gcctgttg Seq ID No: 59 of Bennett et al.SEQ ID NO: 40 gtcttgtttg tgcctgcc Seq ID No: 60 of Bennett et al.SEQ ID NO: 41 caccaccagg gctctcag Seq ID No: 64 of Bennett et al.SEQ ID NO: 42 gggatcacca ccagggct Seq ID No: 65 of Bennett et al.SEQ ID NO: 43 gtcgggaaaa ttgatctc Seq ID No: 71 of Bennett et al.SEQ ID NO: 44 ggagccagga agatcgtc Seq ID No: 73 of Bennett et al.SEQ ID NO: 45 tggagccagg aagatcgt Seq ID No: 74 of Bennett et al.SEQ ID NO: 46 tggcatccat gtaaagtc Seq ID No: 77 of Bennett et al.SEQ ID NO: 47 ggtgcagcct cactgtct Seq ID No: 81 of Bennett et al.SEQ ID NO: 48 aactgcctgt ttgcccac Seq ID No: 82 of Bennett et al.

The following siRNA sequences are suitable for use in the presentinvention. (See, e.g., Pluvinet, et al., Blood, 2004, 104(12),3642-3646), the contents of which are incorporated by reference herein.

(SEQ ID NO: 49): 5_-GCGAAUUCCUAGACACCUGUU-3_ (siRNA-2 of Pluvinet3_-UUCGCUUAAGGAUCUGUGGAC-5_ et al.) (SEQ ID NO: 50):5_-CUGGUGAGUGACUGCACAGUU-3_ (siRNA-6 of Pluvinet3_-UUGACCACUCACUGACGUGUC-5_ et al.) (SEQ ID NO: 51):5_-UACUGCGACCCCAACCUAGUU-3_ (siRNA-8 of Pluvinet3_-UUAUGACGCUGGGGUUGGAUC-5_ et al.)

All siRNA contain a 2 nucleotide overhang at 3′ends.

Oligonucleotides Against Murine CD40

Examples of murine antisense CD40 oligonucleotides are presented below.Further sequence information is found in published patent applicationnumber US 2004/0186071 to Bennett, et al., the contents of which arehereby incorporated by reference herein. The SEQ ID NOS. referred to byBennett, et al. are provided to the right.

Murine

SEQ ID NO: 52 agacaccatc gcag Seq. ID No. 116 of Bennett et al.SEQ ID NO: 53 gcgagatcag aagag Seq. ID No. 117 of Bennett et al.SEQ ID NO: 54 cgctgtcaac aagca Seq. ID No. 118 of Bennett et al.SEQ ID NO: 55 ctgccctaga tggac Seq. ID No. 119 of Bennett et al.SEQ ID NO: 56 ctggctggca caaat Seq. ID No. 120 of Bennett et al.SEQ ID NO: 57 cttgtccagg gataa Seq. ID No. 123 of Bennett et al.SEQ ID NO: 58 cacagatgac attag Seq. ID No. 124 of Bennett et al.SEQ ID NO: 59 tgatatagag aaaca Seq. ID No. 125 of Bennett et al.SEQ ID NO: 60 ctcattatcc tttgg Seq. ID No. 127 of Bennett et al.SEQ ID NO: 61 ggttcagacc agg Seq. ID No. 128 of Bennett et al.SEQ ID NO: 62 tttatttagc cagta Seq. ID No. 130 of Bennett et al.SEQ ID NO: 63 agccccacgc actgg Seq. ID No. 131 of Bennett et al.SEQ ID NO: 64 tctcactcct atcccagt Seq. ID No. 134 of Bennett et al.SEQ ID NO: 65 attagtctga ctcgt Seq. ID No. 138 of Bennett et al.SEQ ID NO: 66 acattagtct gactc Seq. ID No. 139 of Bennett et al.SEQ ID NO: 67 cagatgacat tagtc Seq. ID No. 142 of Bennett et al.SEQ ID NO: 68 ctggactcac cacag Seq. ID No. 143 of Bennett et al.SEQ ID NO: 69 ggactcacca cagat Seq. ID No. 144 of Bennett et al.SEQ ID NO: 70 actcaccaca gatga Seq. ID No. 145 of Bennett et al.SEQ ID NO: 71 tcaccacaga tgaca Seq. ID No. 146 of Bennett et al.SEQ ID NO: 72 accacagatg acatt Seq. ID No. 147 of Bennett et al.SEQ ID NO: 73 agatgacatt ag Seq. ID No. 153 of Bennett et al.SEQ ID NO: 74 cagatgacat tag Seq. ID No. 154 of Bennett et al.SEQ ID NO: 75 acagatgaca ttag Seq. ID No. 155 of Bennett et al.SEQ ID NO: 76 ccacagatga cattag Seq. ID No. 156 of Bennett et al.SEQ ID NO: 77 accacagatg acattag  Seq. ID No. 157 of Bennett et al.SEQ ID NO: 78 caccacagat gacattag Seq. ID No. 158 of Bennett et al.SEQ ID NO: 79 tcaccacaga tgacattag Seq. ID No. 159 of Bennett et al.SEQ ID NO: 80 ctcaccacag atgacattag Seq. ID No. 160 of Bennett et al.

Oligonucleotides Against Rat CD40

Examples of rat antisense CD40 oligonucleotides are presented below.(See, Gao, Ph.D. thesis, 2003, University of Gottingen, Germany).

SEQ ID NO: 81 accgctgtc aacaagcagc (rAS2 of Gao) SEQ ID NO: 82tcctagatggaccgctgt(rAS3 of Gao) SEQ ID NO: 83taacacactgtcctag(rAS4 of Gao)

Oligonucleotides Against Porcine CD40

Examples of porcine antisense CD40 oligonucleotides are presented below.See, Rushworth, et al., Transplantation, 2002, 73(4), 635-642, thecontents of which are incorporated by reference herein.

SEQ ID NO: 84 gctgatgacagtgtttct Aso3 of Rushworth et al.) SEQ ID NO: 85gcctcactctcgctcctg (Aso8 of Rushworth et al.) SEQ ID NO: 86ggactgtatctggactgc (Aso9 of Rushworth et al.) SEQ ID NO: 87gtggacagtcatgtatat (Aso10 of Rushworth et al.)

The present invention therefore provides formulations of amphotericliposomes that exhibit improved stability upon contact with mammalianserum, releasing less or no encapsulated drugs. Such liposomalformulations may be useful in the delivery of drugs after a systemicadministration into the blood stream. The invention especially suits thedelivery of oligonucleotides, a new class of drugs that is currentlyunder development, and DNA plasmids, without being limited to such uses.The majority of such compounds have an intracellular site of action.Carrier systems are used to overcome the poor uptake of such substancesand are sometimes an indispensable prerequisite.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All patents, patent applications, andother references noted herein for whatever reason are specificallyincorporated by reference. The specification and examples should beconsidered exemplary only with the true scope and spirit of theinvention indicated by the following claims.

What is claimed is:
 1. An amphoteric liposome comprising POPC, MoChol,CHEMS, and DOPE, wherein the amphoteric liposome retains at least 60% ofan encapsulated active agent after 4 hours exposure to human serum,wherein MoChol and CHEMS combined comprise 50 to 80 mol % of theamphoteric liposome, wherein POPC and DOPE combined comprise 20 to 50mol % of the amphoteric liposome, wherein POPC, MoChol, CHEMS, and DOPEcombined comprise substantially 100 mol % of the amphoteric liposome,wherein the ratio of DOPE to POPC is from 2 to 4, and wherein the ratioof CHEMS to MoChol is from 0.3 to 0.75.
 2. The amphoteric liposome ofclaim 1, wherein the liposome consists of a formulation selected from:POPC/DOPE/MoChol/CHEMS 6:24:47:23 (mol. %) POPC/DOPE/MoChol/CHEMS10:30:30:30 (mol. %).
 3. The amphoteric liposome of claim 1, wherein theliposome has a size of from 50 to 500 nm.
 4. The amphoteric liposome ofclaim 1, wherein the liposome encapsulates at least one active agent. 5.The amphoteric liposome of claim 4, wherein the active agent comprises anucleic acid.
 6. The amphoteric liposome of claim 4, wherein the activeagent is a circular DNA plasmid, a linear DNA construct, an antisenseoligonucleotide, a decoy oligonucleotide, an agent influencingtranscription, an agent influencing splicing, a ribozyme, a DNAzyme, oran aptamer.
 7. The amphoteric liposome of claim 4, wherein the activeagent is an RNA, an siRNA, an mRNA, an shRNA, or an miRNA.
 8. Theamphoteric liposome of claim 4, wherein the active agent comprises oneor more modified nucleosides selected from locked nucleic acids (LNA),peptide nucleic acids (PNA), 2′O-methyl RNA (2′Ome), or 2′O-methoxyethylRNA (2′MOE) in their phosphate or phosphothioate forms.
 9. Theamphoteric liposome of claim 4, wherein the active agent modulatesexpression of CD40 in mammalian cells.
 10. A pharmaceutical compositioncomprising an effective amount of the active agent-loaded amphotericliposome of claim
 4. 11. A method for treating an inflammatory, immune,or autoimmune disorder in a human in need thereof, the method comprisingadministering to the human a pharmaceutical composition according toclaim
 10. 12. The method of claim 11, wherein the inflammatory, immune,or autoimmune disorder is graft rejection, graft-versus-host disease,diabetes type I, multiple sclerosis, systemic lupus erythematosous,rheumatoid arthritis, asthma, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, psoriasis, thyroiditis, infection, or asolid tumor.
 13. The method of claim 11, wherein the pharmaceuticalcomposition is administered systemically.
 14. The method of claim 11,wherein the pharmaceutical composition is administered locally.